12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 june 2012 to 28 november 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 340 to 382 g (males) and 235 to 291 g (females)
- Fasting period before study:no
- Housing: Females: 5 per cage during pre-pairing then individually housed except during pairing,
Males: 5 per cage except during pairing,
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs or 14 days has elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection analytical method was used.

Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 20 mg/mL and 200 mg/mL. Homogeneity was confirmed following storage at ambient temperature for 2 days and refrigeration for up to 15 days.

Test item concentrations: the test item concentrations in the administered dosage forms analyzed in the first and last weeks of treatment remained within an acceptable range of +5.2% to -2% when compared to the nominal values.

Duration of treatment / exposure:

In the males:
− 2 weeks before mating,
− during the mating period (up to 2 weeks),
− until sacrifice in week 6,

In the females:
− 2 weeks before mating,
− during the mating period (up to 2 weeks),
− during pregnancy,
− during lactation until day 6 post-partum inclusive

Frequency of treatment:

daily

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 13 weeks for males, 13 weeks for females, approximately.

Remarks:

Doses / Concentrations:
0, 100, 300 and 1,000 mg/kg bw/day
Basis:
other: nominal doses

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels used in this study (0, 100, 300 and 1000 mg/kg/day) were selected on the basis of the results of a 4-week toxicity study in the rat by oral route followed by a 2-week recovery period ( Study CVP091/993344). The study concluded that 1000 mg/kg bw/day represented the NOAEL for the test substance.The minor changes noted in some of the parameters measured at this dose level, in the absence of any treatment-related pathological changes or effects on food and bodyweight being observed during the treatment period, were considered not to be of toxicological importance. Based on these observations, a dose-level of 1000 mg/kg bw/day was selected for the high dose. The dose levels for the mid and low doses (300 and 100 mg/kg bw/day respectively) were selected to explore any possible dose relationship.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
-detailed physical examination was performed weekly. F0 females were also examined on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the first day of treatment until pairing. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

For 15 days before pairing, daily vaginal smears were taken from all females. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- The following tests were performed on sperm:
=>Sperm motility: At least 200 sperm were assessed for each male. The percentages of motile and progressively motile sperm were reported for all groups.
=>Sperm morphology: At least 200 sperm were assessed for each male of control and high-dose groups. The percentages of normal sperm and abnormal sperm were reported.
=>Sperm count: The concentration (Million/g) and total number of sperm were reported for each male of control and high-dose groups.
=>Homogenisation-resistant spermatids count: The concentration (Million/g) and total number of spermatids were reported for each male of control and high-dose groups.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
The males were sacrificed during week 5 of treatment. The body and selected organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on the kidneys, liver ,epididymides (only one examined, the other reserved for seminology), prostate, seminal vesicles and testes (only one examined, the other reserved for seminology) from the males in the control- and high dose groups and on all macroscopic lesions.

The dams were sacrificed on day 7 of lactation, the body and selected organs were weighed and a complete macroscopic examination was performed.
A microscopic examination was performed on the kidneys, liver, pituitary,uterus, vagina and ovaries in the control and high-dose groups and on all macroscopic lesions. Mammary area was examined only when premature litter death occured.

GROSS NECROPSY
On completion of the treatment period all surviving F0 males and females were killed by carbon dioxide asphyxiation.
- males: after the end of the pairing period (during week 5 of treatment ),
- females: on day 7 of lactation.,
- females which had not delivered: on day 25 after mating (after a body weight recording to check for a possible un-noticed delivery),
- mothers with litter dying entirely: as appropriate.

For females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique).

Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital:
- surviving pups: on day7 of age.
- Premature deaths if not autolysed or cannibalised.

HISTOPATHOLOGY
A microscopic examination was performed on:
- Liver, kidneys,epididymides, prostate, seminal vesicles, testes, pituitary, uterus, vagina and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period,
- Mammary area of all females with a premature litter death and sacrificed on completion of the treatment period,
- all macroscopic lesions of all the animals sacrificed on completion of the treatment period,

ORGAN WEIGHTS:
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and liver, kidney,epididymides (L and R), prostate, testes (L and R), seminal vesicles and ovaries (L and R) were weighed (wet) as soon as possible after dissection.The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Postmortem examinations (offspring):

SACRIFICE: on day 7 of age

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

- For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.
- For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

# The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For litter data if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
# Sex ratio were analysed by generalised mixed linear model with binomial errors.

Reproductive indices:

Percentage Mating = 100 * (Number animals mating/animals paired)
Fertility index = 100 * (Number animals achieving pregnancy / Number of animals mated)
Gestation index = 100 * (Number of live litters born / Number pregnant)

Offspring viability indices:

- Post-implantation survival index= 100 * (Number of pups born / Number of uterine implantation sites)
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index = 100 * (Number of live born pups on Day 1/ Number of delivered pups)
- Viability index = 100 * (Number of surviving pups on day 7 / Number of live born pups on Day 1)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths and no particular clinical signs.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no effect of treatment on mean bodyweight gain in males and females. and foood consumption of all groups of males receiving the test substance. Food consumption in males treated at 300 or 1000 mg/kg bw/day was marginally high during the two week period before pairing, however the difference was marginal in nature and attributed to normal biological variation and the small study group size. Food consumption in females treated at 100 or 300 mg/kg bw/day before pairing, during gestation and lactation were similar to Control. The marginal difference in food consumption at 1000 mg/kg bw/day throughout gestation, was attributed to lower than expected food consumption in the Control; especially in one female that subsequently had a total litter death.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period showed that the majority of main phase females had regular 4 or 4/5 day oestrous cycles and it was considered that this parameter was not affected by treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- There was no effect of treatment on the weight of testes and epididymides. No adverse effects on sperm motility, concentration or morphology were apparent following treatment at 1000 mg/kg/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights were not affected by treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no findings at macroscopic examination that could be attributed to treatment with the test substance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no findings which were considered to be related to the administration of the test substance. In the seminiferous tubules, no cell or stage specific abnormalities were noted.

OTHER FINDINGS (PARENTAL ANIMALS)
One Control female was killed for welfare reasons following the death of her litter.The animal had an extended parturition and the last surviving pups died before Day 1 post-partum recordings were made.

Dose descriptor:

NOEL

Remarks:

Reproductive performance

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

The assessment of litter responses detailed below is made based on the 9, 10, 9 and 10 litters in each of groups 0, 100, 300 and 1000 mg/kg bw/day, respectively.

VIABILITY (OFFSPRING)
There was no effect of parental treatment with the test substance on mean number of implantations, live litter size on Day 1 and offspring survival up to Day 7 of age.

CLINICAL SIGNS (OFFSPRING)
The general appearance and behaviour of the offspring were not affected by maternal treatment.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight on Day 1 of age was not affected by parental treatment. Subsequent bodyweight gain up to Day 7 of age appeared to be marginally higher in offspring with maternal treatment at 1000 mg/kg/day.


GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormalities.

OTHER FINDINGS (OFFSPRING):
There was no effect of parental treatment with the test substance on sex ratio.

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Conclusions:

It was conclude that oral administration of the test substance to Sprague-Dawley rats for at least four weeks at doses of 100, 300 and 1000 mg/kg/day was well tolerated with no toxicologically significant systemic effects and there was no effect of treatment on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.

Executive summary:

In a screening study for reproductive / developmental effects performed according to OECD 421 , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum.

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of four consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 

During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults.  Oestrous cycles and gestation length were assessed and parturition observations were performed for females.  Sperm analysis and sperm morphology were performed for males.The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

Overall treatment with the test substance was well-tolerated at all doses, with no adverse effect on general condition or signs following administration related to treatment. Bodyweight gain was unaffected by treatment. There were some marginal intergroup variations in food consumption which were attributed to normal biological variation and the small study group size, as the differences were marginal in nature and there was no effect on bodyweight gain. In addition, one animal in the Control group was humanely killed following the death of her litter and data showed that this animal had low food consumption, when compared to other animals in the Control group and an extended parturition.

There was no effect of treatment on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length, gestation index; litter size, offspring survival and sex ratio or offspring bodyweight gain or clinical signs to Day 7 of age. There were no macropathological findings in the adults or offspring and no difference in adult organ weights or sperm analysis/ morphology. Histopathological examination revealed no change in any observed tissues or change in seminiferous tubules of the testes with respect to spermatogenic cycle and the integrity of the various cell types present within the different stages.

It was concluded that the NOEL for the test substance was 1000 mg/kg bw/day (the limit dose) for reproductive performance and offspring growth and survival. For the general toxicity, the NOAEL was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Screening reprotoxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a screening study for reproductive / developmental effects performed according to OECD 421 and in compliance with Good Laboratory Practice, the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum.

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of four consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 

During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults.  Oestrous cycles and gestation length were assessed and parturition observations were performed for females. Sperm analysis and sperm morphology were performed for males.The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

Overall treatment with the test substance was well-tolerated at all doses, with no adverse effect on general condition or signs following administration related to treatment. Bodyweight gain was unaffected by treatment. There were some marginal intergroup variations in food consumption which were attributed to normal biological variation and the small study group size, as the differences were marginal in nature and there was no effect on bodyweight gain. In addition, one animal in the Control group was humanely killed following the death of her litter and data showed that this animal had low food consumption, when compared to other animals in the Control group and an extended parturition.

There was no effect of treatment on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length, gestation index; litter size, offspring survival and sex ratio or offspring bodyweight gain or clinical signs to Day 7 of age. There were no macropathological findings in the adults or offspring and no difference in adult organ weights or sperm analysis/ morphology. Histopathological examination revealed no change in any observed tissues or change in seminiferous tubules of the testes with respect to spermatogenic cycle and the integrity of the various cell types present within the different stages.

It was concluded that the NOEL for the test substance was 1000 mg/kg bw/day (the limit dose) for reproductive performance and offspring growth and survival. For the general toxicity, the NOAEL was considered to be 1000 mg/kg bw/day.

Short description of key information:
In a study performed according to the OECD 421 (Leggett, 2012), the substance was administered daily by oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOEL for the substance was 1000 mg/kg bw/day (the limit dose) for reproductive performance and offspring growth and survival. For general toxicity, the NOAEL was considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Study performed according to the OECD guideline 421 and GLP compliant.

Effects on developmental toxicity

Description of key information

In a study performed according to the OECD 414 (Leggett, 2013), the substance was administered daily by oral administration (gavage) to pregnant female rats from Day 6 to Day 19 post-coitum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for  the substance was 1000 mg/kg bw/day (the limit dose) for both maternal toxicity and embryo-fetal development.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 june 2012 to 8 february 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 228 to 286 g at the start of the study (Day 0 of gestation)
- Fasting period before study:no
- Housing:Up to 4 per cage during acclimation then individually housed except during pairing,
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 5 days before they were paired on a 1:1 basis with stock males from the same source.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection analytical method was used.

Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 20 mg/mL and 200 mg/mL. Homogeneity was confirmed following storage at ambient temperature for 2 days and refrigeration for up to 15 days.

Test item concentrations: the test item concentrations in the administered dosage forms analyzed in the first and last weeks of treatment were within +/- 10% of nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Only females with a sperm positive vaginal smear or at least two copulation plugs were selected.

Duration of treatment / exposure:

From Day 6 to Day 19 (inclusive) after mating

Frequency of treatment:

Daily

Duration of test:

Sacrifice on Day 20 after mating

Remarks:

Doses / Concentrations:
0, 100, 300 and 1,000 mg/kg bw/day
Basis:
other: nominal doses

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The doses were selected on the basis of the results of the reproduction/development toxicity screening test (Huntingdon Life Sciences Report Number: FIN0048). In that study, there was no maternal or reproductive/developmental toxicity.

- Rationale for animal assignment (if not random): Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries

OTHER: no

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses recorded for each uterine horn

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

# The following sequence of statistical tests was used for bodyweight, gravid uterine weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests were made. For all other comparisons, the F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

# For corpora lutea, implantations, live young, fetal, placental and litter weight data,if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

# Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors.

Indices:

Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova. It was calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations)/ Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero.

Post-implantation loss was considered to exclude the first two to three days post-implantation as deaths occurring at this stage are considered to leave no remains visible at Day 20 of gestation. It was calculated from the formula:
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/Number of implantations] x 100

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- There were no clinical signs and no effect of treatment on bodyweight gain.
- The food consumption of females receiving 1000 mg/kg/day was statistically high, when compared with Control. However this intergroup difference was small and these females had eaten marginally more than Control before commencement of treatment, therefore no toxicological significance is attached to this finding.
- Macroscopic examination of females on Day 20 of gestation did not reveal any findings that were considered to be related to treatment.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- All females were pregnant. Therefore, the assessment was based on the 22 females with live young in the Control group and each treatment group at termination, on Day 20 of gestation:
Litter data as assessed by mean corpora lutea, implantations, early, late and total resorption counts, live young, sex ratio, and pre- and post-implantation loss, for females receiving 100, 300 or 1000 mg/kg/day was similar to Control and considered unaffected by treatment.

- Mean male and female fetal weights were marginally higher than Control following maternal treatment at 1000 mg/kg/day and was associated with marginally higher overall mean fetal weight. However, as the range of individual fetal weights was similar to Control, the difference was considered fortuitous and not biologically relevant or toxicologically important. Litter size, litter or placental weights were considered unaffected by treatment.

- The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The repeated daily oral administration of the test substance to pregnant Sprague-Dawley rats from Day 6 to 19 of gestation at doses of 100, 300 or 1000 mg/kg bw/day was well tolerated. It was therefore concluded that 1000 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development.

Executive summary:

In a prenatal development toxicity study performed according to OECD 414 , the objective was to evaluate the potential toxic effects of the test substance on the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 19 post-coitum, inclusive. 

Three groups of 22 female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 of gestation and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral or skeletal examination.

Maternal clinical condition, bodyweight, food consumption, macropathology, pregnancy outcome, embryo-fetal survival, growth and development were unaffected by maternal treatment at doses up to and including 1000 mg/kg/day.

It was therefore concluded that 1000 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Developmental toxicity study complete and sufficient to fulfill the REACh annex IX requirements

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal development toxicity study performed according to OECD 414 and in compliance with Good Laboratory Practice, the objective was to evaluate the potential toxic effects of the test substanceon the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 19 post-coitum, inclusive. 

Three groups of 22 female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 of gestation and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral or skeletal examination.

Treatment with the test substance was well tolerated at all doses. Maternal clinical condition, bodyweight, food consumption, macropathology, pregnancy outcome, embryo-fetal survival, growth and development were unaffected by maternal treatment at doses up to and including 1000 mg/kg bw/day.

It was therefore concluded that 1000 mg/kg bw/day ( the limit dose) was the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development.

Justification for selection of Effect on developmental toxicity: via oral route:
Study performed according to the OECD guideline 414 and GLP compliant.

Justification for classification or non-classification

No effects on reproductive performance and offspring growth and survival were observed in a Reproduction / Developmental Toxicity Screening Test at the limit dose of 1000 mg/kg bw/day.

No maternal toxicity and no effects on embryo-fetal survival, growth and development were observed in a Prenatal/ developmental toxicity study at the limit dose of 1000 mg/kg bw/day.

On the basis of these results and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to toxicity to reproduction.